Total IgE in tears accurately reflects the severity and predicts the prognosis of seasonal allergic conjunctivitis

Abstract Background Although immunoglobulin E (IgE) increases significantly in tears and serum during seasonal allergic conjunctivitis (SAC), it is unclear whether tear total IgE can reflect the severity and prognosis of SAC more accurately than serum total IgE. We aimed to investigate the usefulness of measuring the total IgE in tears to evaluate the severity and determine the treatment of SAC. Methods This prospective, nonrandomized study involved 55 patients with SAC and 10 age‐ and sex‐matched healthy controls. Serum and tears were collected before and after treatment to analyze the total IgE. SAC patients received the same topical anti‐allergy treatment and were followed‐up every 2 weeks for 1 month. The relationship of tear and serum total IgE concentrations with pollen concentrations and symptom severity before and after treatment was assessed. Results The total IgE concentration in tears was higher in SAC patients than in healthy participants with significant correlations between tear and serum total IgE concentrations. The total IgE concentration in tears, but not in serum, correlated with the pollen concentration and severity of ocular symptoms and reactions in SAC. Treatment‐associated improvements in symptoms and reactions in SAC correlated with decreased concentrations of the tear total IgE. Patients with disease recurrence following treatment demonstrated significantly higher tear total IgE concentrations than patients with no recurrence. Conclusion The total tear IgE level can indicate the severity and predict the prognosis of SAC more accurately than the serum total IgE.

Since its discovery more than 50 years ago, immunoglobulin E (IgE) has been shown to be involved in a wide variety of immunological diseases, including allergic disease. [9][10][11] Despite being the least abundant immunoglobulin in normal human serum, IgE plays a key role in SAC. 12,13 IgE, when secreted in tears, binds to fragment crystallizable (Fc) receptors on mast cells. 14 In the case of re-exposure to allergens, it leads to mast cell degranulation release of mediators such as histamine, resulting in chemosis and ocular itching.
The diagnosis of ocular allergies is primarily clinical, although laboratory tests can be useful in supporting the diagnosis. 6 At present, laboratory tests for AC mainly include cytological examination, skin prick tests, and total IgE antibody detection. [15][16][17] The detection of eosinophils is a complex procedure with a low detection rate of approximately 60% in conjunctival scrapings from patients with mild allergic conditions. 18 Skin prick tests are invasive and require multiple pricks with small volumes of allergens. 8 Owing to the large ocular surface reaction caused by the conjunctival provocation test, the clinical application of this test is limited. 19,20 Moreover, although serum total IgE tests are recommended for etiological diagnosis, the test does not provide information on the pathogenesis and severity of the disease. 21 Given its high prevalence, it is therefore important to measure the total IgE in tears to help diagnose SAC. 22 However, assessing the severity of SAC by measuring the total IgE in tears remains to be elucidated. We hypothesized that determining the total IgE levels in tears in patients with SAC, relative to the serum total IgE, may allow for better evaluation of the severity of SAC and allow for prediction of the prognosis of the disease.
Thus, the aim of this study was to explore whether the total IgE levels in tears or serum can better reflect the severity of the disease in providing a baseline for the diagnosis and treatment of SAC.

| Study design
This was a prospective, non-randomized study conducted at the

| Participants
Fifty-five patients (19 men and 36 women; mean age: 31 years; age range: 7-56 years ( Figure 1A)) with SAC were enrolled in the study from March 2020 to August 2020 ( with a history of cataract surgery, corneal refractive surgery, or infectious conjunctivitis were also excluded, as were patients with other allergic diseases, including allergic rhinitis, allergic asthma, and allergic dermatitis. 23,24 Patients over 60 years and under 5 years old were also excluded. Participants with chronic diseases, mental disorders, on medication, or who abused drugs were excluded. Furthermore, participants on continuous drugs for a chronic disease or smoking as well as those who had parasitic infections were also excluded. In addition, 10 healthy age-and sex-matched participants with no history of allergic diseases were recruited as the healthy control group.

| Measurement of the pollen concentration
Pollen concentrations in areas of patients' residence were recorded using the Durham pollen collector, 25 which was placed 14-16 m above from the ground. The sampling slides were replaced every 24 h, and the collected samples were stained with alkaline fuchsine before microscopic analysis. The unit for pollen concentration was grains per thousand square millimeters.

| Diagnosis of SAC
The diagnosis of AC was based on the results of a slit-lamp examination and an allergen test. The diagnosis was made by an ophthalmologist based on published guidelines, and successful treatment of conjunctivitis was noted when a patient reported the symptom of ocular itching to have disappeared or to occur only occasionally (score = 1) which was judged to be significantly improved.

| Symptom and sign scores of allergic conjunctivitis
All participants completed a questionnaire at the first visit and at each follow-up visit, and their symptoms were scored with a doctor's assistance. The symptom scores were obtained for both eyes for each patient; however, for the purpose of this study, only the score from the right eye was used for analysis. The doctor who assisted patients with their symptom score also scored the ophthalmic results of each patient's slit-lamp examination (Supplemental Table S1). [26][27][28][29][30]

| Measurement of total IgE in tears
Three microliters of tears were collected from each patient using a capillary pipette at the first visit and at each of the follow-up visits.   The lateral flow immunoassay was utilized to quantitatively determine the amount of human IgE in the tears. Nonstimulated tear fluids from the lateral canthus of one eye were collected using a disposable microcapillary fluid collector (3 μL; Seninda Biomedical Corporation, China); the tear fluids were stored in a 0.5 ml centrifuge tube, and the sample was frozen immediately at −80°C. A total of 2.2 μL of the sample was dispensed into the test card (Seninda Biomedical Corporation, China), and three full drops of buffer were added. The test card was allowed to rest undisturbed for 10 min, and then the i-ImmunDx Analyzer was used for reading. The test was performed separately for each eye.

| Determination of the serum total IgE
A total of 2 ml of blood was collected from each patient at the first visit. The serum was separated and stored by freezing at −80°C until it was analyzed. The serum total IgE concentration in the serum sample was detected using an ELISA kit (BMS2097, Thermo Fisher, USA) and quantified according to the manufacturer's instructions.

| Treatment and follow-up
All patients received the same topical anti-allergy treatment. The drugs used were fluorometholone eye drops, olopatadine eye drops, and sodium hyaluronate eye drops. All drugs were used according to the manufacturer's instructions. After the first visit, the participants were instructed to return to the consulting room for two further visits (one visit every 2 weeks). Thereafter, they were followed-up via three telephone consultations every other month. Recurrence was defined as recurrence of the disease during the follow-up period.

| Statistical analysis
GraphPad 8.0 software and SPSS 18.0 software were used for analysis and mapping. The differences between groups were compared using the two-tailed Mann-Whitney U test. Any associations between variables were calculated using Spearman's correlation coefficient. The level of significance was set at p < 0.05. Data are presented as the mean � standard error of the mean (SEM).

| Levels of IgE in tears and serum increased in SAC
Fifty-three of the 55 participants with SAC attended the two followups, during which photographs of the surface of the eyes were taken for further assessment. The baseline concentrations of total IgE in tears and in the serum of patients with SAC were significantly higher than those of control participants (p < 0.01; Figure 1B). Spearman's correlation analysis showed that serum total IgE positively correlated with tear total IgE (r = 0.44, p < 0.01; Figure 1C). Furthermore, there was a significant correlation between pollen concentration and the total IgE in tears (r = 0.55, p < 0.01) but not between pollen concentration and serum total IgE (r = 0.26, p = 0.06; Figure 1D).

| Total IgE in tears positively correlated with the severity of symptoms
Assessment of the correlation between the total IgE or serum total IgE concentration and the ocular itch score revealed that the total IgE concentration in tears, but not the serum total IgE concentration, was significantly positively correlated with the itch score ( Figure 2A).
Similar results were also noted for correlations between the total IgE or serum total IgE concentration and tearing score, redness score, foreign body sensation score, and burning sensation score

| IgE in tears positively correlated with the severity of signs
The total IgE concentration in tears, but not the serum total IgE concentration, was also found to be significantly increased with an increase in palpebral conjunctiva hyperemia score ( Figure 3A).
While significant correlations were noted between total IgE concentrations in tears and the palpebral conjunctiva swelling score, bulbar conjunctiva hyperemia score, bulbar conjunctiva chemosis score, palpebral conjunctiva follicle score, and palpebral conjunctiva papillae score, the serum total IgE concentration showed a significant difference only in patients with severe disease and in patients without the apparent conjunctival reaction ( Figure 3B-F).

| Tear IgE correlated with the speed of improvement and recurrence of SAC
Patients were separated into two groups, those who improved within 7 days and those who improved after 7 days. The total IgE in the tears of patients who improved within treatment for 7 days was significantly lower than that in the tears of the patients who improved after treatment for 7 days (p < 0.01; Figure 4A). In contrast, there was no significant difference in the serum total IgE concentration between the two groups. Spearman's correlation analysis further demonstrated that the total IgE concentration in tears before treatment positively correlated with the number of days until symptom improvement (p = 0.86; Figure 4A). Assessment of the total IgE in tears and serum of patients who had recovered or relapsed following treatment showed that total IgE concentrations in tears at the initial visit were significantly higher in patients who relapsed after drug discontinuation than in patients who had recovered (p = 0.03), whereas serum total IgE concentrations were not significantly different between the two groups (p = 0.81; Figure 4B).
A typical case is shown in Figure 4.

| The change in tear IgE can reflect the evolution of disease severity
In this study, 53 participants completed two follow-up visits. The results showed that with patient improvement, the concentration of  Among these cytokines, interleukin 4 (IL-4) promotes the synthesis of total IgE, whereas interferon-gamma (IFN-γ) inhibits IL-4-induced total IgE. 35,36 However, the correlation coefficient for tear total IgE and pollen concentrations was relatively low (r = 0.55). It is possible that this is because we analyzed the total IgE in tears and not specifically pollen IgE. Recently, Yamana et al. 37 also found that pollen-induced AC can be caused by pollen sensitization in the conjunctiva since pollenspecific IgE antibodies have a higher total tear IgE positive rate than other antigens. This further confirms our results. Nonetheless, we will focus more on tear-specific IgE in future studies.  Indeed, this measurement has several advantages over measurement of the serum total IgE. In particular, immunochromatography and double antibody Sandwich method are quicker to perform than the standard conventional method and, therefore, may be of considerable benefit to the patient, as SAC may be diagnosed at the patient's first visit. Furthermore, this is a non-invasive technique that requires only 3 μL of tears to diagnose SAC. Compared with serum, tears also have better stability, are easy to transport and store, and have a specific field application value. Additionally, compared with that of serum total IgE, sensitivity of tear total IgE is higher and can be detected at a low dose and used in micro amounts. Another simple and fast technology called "Allerwatch" can also detect total IgE levels in AC. 45,46 However, at present, such testing products cannot be purchased in China. "Allerwatch" is a qualitative product, which that can only be used to judge a negative and positive diagnoses of AC. Seinda's i-immudx IgE test is a quantitative product, that can detect the concentration of IgE in tears. Therefore, it can be used not only for auxiliary diagnosis, but also for evaluating the curative effect.
One of the limitations of this study was that we excluded patients with atopic keratoconjunctivitis because the study focused on SAC. In the future, we will study the relationship between the total tear IgE levels and specific serum IgE levels in patients with atopic keratoconjunctivitis. Further, considering the favorable principle of bioethics, we only collected serum samples of the participants at the first visit to detect the serum total IgE and did not measure its dynamic change. Furthermore, in line with the principle of bioethics, only10 healthy volunteers were included in our study. In future research, we will pay more attention to the consistency of the numbers of the control and experimental groups. Moreover, all included patients had mild to moderate SAC. Therefore, further evaluation is needed for patients with severe AC. BAO ET AL.